The invention relates to methods and compositions for the recombinant production of Onc, a cytotoxic ribonucleolytic protein having anti-tumor and anti-viral properties. In particular, the invention relates to a recombinant Onc protein having an amino terminal methionine and comprising an Onc polypeptide.
ONCONASE, or Onc, is a ribonuclease purified from Rana pipiens oocytes. While Onc is homologous to pancreatic RNases in amino acid sequence (Ardelt et al., J. Biol. Chem. 266:245-251 (1991)) and three dimensional structure (Mosimann et al., J. Mol. Biol. 236:1141-1153 (1994)), its pharmacological properties are quite unique Onc displays cytostatic and cytotoxic activity against numerous cancer cell lines in vitro (Darzynkiewicz et al., Cell Tissue Kinet. 21: 169-182 (1988)), is up to five-thousand times more toxic to animals than is the homologous protein, RNase A (Newton et al., J. Neurosci. 14(2):538-44 (1994)), and displays anti-tumor activity in vivo (Mikulski et al., J. Natl. Canc. Inst. 82:151-152 (1990); Int. J. Oncol. 3:57-64 (1993). Moreover, Onc has been found to specifically inhibit HIV-1 replication in infected H9 leukemia cells at non-cytotoxic concentrations (Youle et al., Proc. Natl. Acad. Sci. USA 91:6012-6016 (1994)). Such promising pharmacologic properties explain why this protein is currently the subject of phase III clinical trials.
Unfortunately, since Onc is isolated from oocytes, procurement of an adequate supply is uncertain. Recent concerns regarding the availability of the anti-cancer compound taxol illustrate some of the problems of obtaining natural products for use as pharmaceuticals. Similarly, availability of Onc is increasingly problematic in light of the declining population of R. pipiens and the seasonal variation in the supply of its oocytes.
Accordingly, what is needed in the art is a means to produce Onc by recombinant methods so as to meet demand for this therapeutic and alleviate the impact on its native source. Further, what is needed is a means to derivatize and alter the sequence of Onc to provide more efficacious compounds. Quite surprisingly, the present invention provides these and other advantages.
The present invention is directed to an rOnc protein, comprising a polypeptide of SEQ ID NO:1 or conservatively modified variant thereof. Preferably, the polypeptides of the invention have a glutamine residue at position +1 of the polypeptide. Even more preferably, the glutamine residue is at the amino terminus of the rOnc protein.
In one embodiment, the polypeptide comprises a hydrophobic residue at position 23. In a further embodiment, the polypeptide comprises the amino acid leucine at position. 23. Preferably, the polypeptides of the present invention also have a lysine at position 9, a histidine at position 10, a histidine at position 97, a lysine at position 31, a phenylalanine at position 98, and a threonine at position 35.
In another embodiment, the amino acid sequence of rOnc protein is generally modified so that it is not susceptible to cleavage by cyanogen bromide. Preferably, upon cyclization of the amino terminal glutamine to pyroglutamyl, the polypeptide of SEQ ID NO:1 or conservative variants thereof have a relative IC50 in U251 cells at least 50% that of the polypeptide of SEQ ID NO:2. Polypeptides of the present invention may be joined to a ligand binding moiety such as an immunoglobulin.
In another aspect of the present invention, a rOnc protein is provided. The rOnc protein comprises a polypeptide of SEQ ID NO:1 or conservatively modified variant thereof, preferably with a glutamine residue at position 1, and an amino terminal methionine. Nucleic acids encoding for the rOnc protein of the present invention are also provided. In a preferred embodiment, the amino terminal methionine is directly linked to the polypeptides of the present invention. The amino terminal methionine may also be linked to the polypeptides of the present invention via less than 50 amino acid residues.
In another aspect of the present invention, a method of making a rOnc protein is provided. The method comprises expressing in a host cell a nucleic acid encoding a rOnc protein comprising a polypeptide of SEQ ID NO:1 or conservatively modified variant thereof and an amino terminal methionine; cleaving the amino terminal methionine with a cleaving agent; and causing the glutamine residue at position 1 of SEQ ID NO:1 or conservative variant thereof to cyclize to a pyroglutamyl residue. In one embodiment, the nucleic acid encodes a hydrophobic residue at position 23 of the polypeptide. Preferably, the nucleic acid encodes a leucine at position 23 when the cleaving agent is cyanogen bromide. The cleaving agent is typically a peptidase or cyanogen bromide.
In another aspect of the present invention, a host cell is provided that expresses a nucleic acid coding for an rOnc protein. The rOnc protein comprises a polypeptide of SEQ ID NO:1 or conservatively modified variant thereof, and wherein the polypeptide has a glutamine at position 1; and an amino terminal methionine. An expression vector encoding a polypeptide of SEQ ID NO:1 or conservatively modified variant thereof, wherein the polypeptide has a glutamine at position 1, and an amino terminal methionine is also provided. In one embodiment, the expression vector encodes a leucine at position 23 of the polypeptide. In another, the expression vector encodes methionine or another hydrophobic residue at position 23 of the polypeptide.
The present invention has utility in providing a means to recombinantly produce rOnc for use as an anti-cancer, anti-tumor, and anti-viral composition. Additionally, the rOnc proteins of the present invention also have use as a cell culture selection agent against cancerous or tumorigenic cells thereby providing, for example, a means to select and identify gene therapy compositions which inhibit tumorigenic growth.
The present invention is directed to recombinant Onc (rOnc), a potent anti-tumor and anti-viral compound derived from P-30 Protein, an oocyte protein of Rana pipiens (Ardelt et al., J. Biol. Chem. 266:245-251 (1991)) and exemplified by the product ONCONASE, a registered tradename of the Alfacell Corporation, Bloomfield, N.J. The invention provides, inter alia, rOnc compositions, and compositions and methods for making rOnc.
The rOnc polypeptide of the present invention is altered in amino acid sequence relative to the native P-30 Protein (nOnc) such that recombinant production and subsequent conversion of this protein to its pharmacologically active form is readily achieved. Recombinant Onc has in vivo and in vitro utility. Recombinant Onc may be employed as an anti-cancer, anti-tumor, and anti-viral composition, or, for example, as a selection agent in cell culture work against tumorigenic cells. Thus, in some embodiments rOnc may be employed to inhibit HIV-1 replication, or to treat pancreatic cancer.
In contrast to homologous RNases, rOnc lacks appreciable ribonuclease activity or cytotoxicity when expressed with a methionine residue at the amino terminus. When chimeric proteins composed of rOnc and human pancreatic RNase (hRNase) sequences are constructed, they yield enzymes with similar substrate specificity and activity to that of Onc; however, they too lack appreciable cytoxicity. Thus, the features lending cytotoxicity to rOnc were not readily discernible. We have discovered that the amino terminal pyroglutamyl residue of rOnc plays a part in the cytotoxicity of this enzyme. Our identification of the role of this residue, and means to modify Onc for recombinant production while maintaining the desired cytotoxic activity provides, in part, the invention as described herein.
Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by%one of ordinary skill in the art to which this invention belongs. Singleton et al. (1994) Dictionary of Microbiology and Molecular Biology, second edition, John Wiley and Sons (N.Y.), and Hale and Marham (1991) The Harper Collins dictionary of Biology, Harper Perennial, NY provide one of skill with a general dictionary of many of the terms used in this invention. Although any methods and materials Similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described. For purposes of the present invention, the following terms are defined below.
Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.
xe2x80x9cConservatively modified variantsxe2x80x9d applies to both amino acid and nucleic acid-sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given polypeptide. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are xe2x80x9csilent variations,xe2x80x9d which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid which encodes a polypeptide is implicit in each described sequence.
As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a xe2x80x9cconservatively modified variantxe2x80x9d where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Conservative modifications also include the deletion of 1, 2, 3, 4, 5, 6, or 7 amino acids from the carboxy end of SEQ ID NO:1 (extending to Histidine at position 97).
The xe2x80x9cconservatively modified variantsxe2x80x9d of the polypeptides of the present invention are cytotoxic, as defined below, or alternatively, the xe2x80x9cconservatively modified variantsxe2x80x9d are capable of forming a cytotoxic compound as a result of formation of an amino terminal pyroglutamyl residue. Those of skill will recognize that when the glutamine residue is the amino-terminal group in a peptide, polypeptide, or protein, it tends to spontaneously cyclize to pyrrolidone carboxylic acid (i.e., xe2x80x9cpyroglutamylxe2x80x9d) which is a particularly preferred form of the amino terminal glutamine residue.
The following six groups each contain amino acids that are conservative substitutions for one another:
1) Alanine (A), Serine (S), Threonine (T);
2) Aspartic acid (D), Glutamic acid (E);
3) Asparagine (N), Glutamine (Q);
4) Arginine (R), Lysine (K);
5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and
6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W). See also, Creighton (1984) Proteins W. H. Freeman and Company.
The terms xe2x80x9cisolatedxe2x80x9d or xe2x80x9cbiologically purexe2x80x9d refer to material which is substantially or essentially free from components which normally accompany it as found in its naturally occurring environment. The isolated material optionally comprises material not found with the material in its natural environment. The rOnc proteins described herein are isolated and biologically pure since they are recombinantly produced in the absence of unrelated Rana pipiens proteins. They may, however, include heterologous cell components, a ligand binding moiety, a label and the like.
The term xe2x80x9cnucleic acidxe2x80x9d refers to a deoxyribonucleotide or ribonucleotide polymer in either single- or double-stranded form, and unless otherwise limited, encompasses known analogues of natural nucleotides that hybridize to nucleic acids in manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence includes the complementary sequence thereof. A nucleic acid encodes another nucleic acid where it is the same as the specified nucleic acid, or complementary to the specified nucleic acid.
An xe2x80x9cexpression vectorxe2x80x9d includes a recombinant expression cassette which includes a nucleic acid which encodes a rOnc protein which can be transcribed and translated by a cell. A recombinant expression cassette is a nucleic acid construct, generated recombinantly or synthetically, with a series of specified nucleic acid elements which permit transcription of a particular nucleic acid in a target cell. The expression vector can be part of a plasmid, virus, or nucleic acid fragment. Typically, the recombinant expression cassette portion of the expression vector includes a nucleic acid to be transcribed, and a promoter.
The term xe2x80x9crecombinantxe2x80x9d when used with reference to a protein indicates that a cell expresses a peptide, polypeptide, or protein (collectively xe2x80x9cproteinxe2x80x9d) encoded by a nucleic acid whose origin is exogenous to the cell. Recombinant cells can express genes that are not found within the native (non-recombinant) form of the cell. Recombinant cells can also express genes found in the native form of the cell wherein the genes are reintroduced into the cell by artificial means, for example under the control of a heterologous promoter.
The term xe2x80x9csubsequencexe2x80x9d in the context of a particular nucleic acid or polypeptide sequence refers to a region of the nucleic acid or polypeptide equal to or smaller than the particular nucleic acid or polypeptide.
xe2x80x9cStringent hybridization wash conditionsxe2x80x9d in the context of nucleic acid hybridization experiments such as Southern and northern hybridizations are sequence dependent, and are different under different environmental parameters. An extensive guide to the hybridization of nucleic acids is found in Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biologyxe2x80x94Hybridization with Nucleic Acid Probes Part I, Chapter 2 xe2x80x9cOverview of principles of hybridization and the strategy of nucleic acid probe assaysxe2x80x9d, Elsevier, N.Y. Generally, highly stringent wash conditions are selected to be about 5xc2x0 C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Very stringent conditions are selected to be equal to the Tm point for a particular probe. Nucleic acids which do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides which they en code are substantially identical. This occurs, e.g., when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code.
The term xe2x80x9cidenticalxe2x80x9d in the context of two nucleic acid or polypeptide sequences refers to the residues in the two sequences which are the same when aligned for maximum correspondence over a specified comparison window. When percentage of sequence identity is used in reference to proteins or peptides it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g. charge or hydrophobicity) and therefore do not change the functional properties of the molecule. Where sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well-known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated, e.g., according to the algorithm of Meyers and Miller, Computer Applic. Biol. Sci., 4: 11-17 (1988) e.g., as implemented in the program PC/GENE (Intelligenetics, Mountain View, Calif., USA).
Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman (1981) Adv. Appl. Math. 2: 482; by the homology alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48: 443; by the search for similarity method of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85: 2444; by computerized implementations of these algorithms (including, but not limited to CLUSTAL in the PC/Gene program by Intelligenetics, Mountain View, Calif., GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis., USA); the CLUSTAL program is well described by Higgins and Sharp (1988) Gene, 73: 237-244 and Higgins and Sharp (1989) CABIOS 5: 151-153; Corpet, et al. (1988) Nucleic Acids Research 16, 10881-90; Huang, et al. (1992) Computer Applications in the Biosciences 8, 155-65, and Pearson, et al. (1994) Methods in Molecular Biology 24, 307-31. Alignment is also often performed by inspection and manual alignment.
The term xe2x80x9csubstantial identityxe2x80x9d orxe2x80x9csubstantial similarityxe2x80x9d in the context of a polypeptide indicates that a polypeptide comprises a sequence with at least 70% sequence identity to a reference sequence, or preferably 80%, or more preferably 85% sequence identity to the reference sequence, or most preferably 90% identity over a comparison window of about 10-20 amino acid residues. An indication that two polypeptide sequences are substantially identical is that one peptide is immunologically reactive with antibodies raised against the second peptide. Thus, a polypeptide is substantially identical to a second polypeptide, for example, where the two peptides differ only by a conservative substitution.
One indication that two nucleic acid sequences are substantially identical is that the polypeptide which the first nucleic acid encodes is immunologically cross reactive with the polypeptide encoded by the second nucleic acid.
Another indication that two nucleic acid sequences are substantially identical is that the two molecules hybridize to each other under stringent conditions. Stringent conditions are sequence dependent and are different under different environmental parameters. Generally, stringent conditions are selected to be about 5xc2x0 C. to 20xc2x0 C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. However, nucleic acids which do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides which they encode are substantially identical. This occurs, e.g., when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code.
The term xe2x80x9cspecifically deliverxe2x80x9d as used herein refers to the preferential association of a molecule with a cell or tissue bearing a particular target molecule or marker and not to cells or tissues lacking that target molecule. It is, of course, recognized that a certain degree of non-specific interaction may occur between a molecule and a non-target cell or tissue. Nevertheless, specific delivery, may be distinguished as mediated through specific recognition of the target molecule. Typically specific delivery results in a much stronger association between the delivered molecule and cells bearing the target molecule than between the delivered molecule and cells lacking the target molecule. Specific delivery typically results in greater than 2 fold, preferably greater than 5 fold, more preferably greater than 10 fold and most preferably greater than 100 fold increase in amount of delivered molecule (per unit time) to a cell or tissue bearing the target molecule as compared to a cell or tissue lacking the target molecule or marker.
The term xe2x80x9cresiduexe2x80x9d as used herein refers to an amino acid that is incorporated into a polypeptide. The amino acid may be a naturally occurring amino acid and, unless otherwise limited, may encompass known analogs of natural amino acids that can function in a similar manner as naturally occurring amino acids.
A xe2x80x9cfusion proteinxe2x80x9d or when a molecule is xe2x80x9cjoinedxe2x80x9d to another refers to a chimeric molecule formed by the joining of two or more polypeptides through a peptide bond formed between the amino terminus of one polypeptide and the carboxyl terminus of another polypeptide. The fusion protein or the joined molecules may be formed by the chemical coupling of the constituent molecules or it may be expressed as a single polypeptide from a nucleic acid sequence encoding a single contiguous fusion protein. A single chain fusion protein is a fusion protein having a single contiguous polypeptide backbone.
A xe2x80x9cligandxe2x80x9d or a xe2x80x9cligand binding moietyxe2x80x9d, as used herein, refers generally to all molecules capable of specifically delivering a molecule, reacting with or otherwise recognizing or binding to a receptor on a target cell. Specifically, examples of ligands include, but are not limited to, immunoglobulins or binding fragments thereof, lymphokines, cytokines, receptor proteins such as CD4 and CD8, solubilized receptor proteins such as soluble CD4, hormones, growth factors such as epidermal growth factor (EGF), and the like which specifically bind desired target cells.
xe2x80x9cCytotoxicityxe2x80x9d, as used herein, refers to the inhibition of protein synthesis in NIH 3T3 (ATCC No. CRL 1658) cells using the protocol described in Wu et al., J. Biol. Chem. 270:17476-17481 (1995). A cytotoxic protein of the present invention will have a relative 50% inhibitory concentration (IC50) at least 20% that of an equimolar amount of the polypeptide of SEQ ID NO:2. More preferably, the relative IC50 will be at least 30% or 40% that of the polypeptide of SEQ ID NO:2, and even more preferably, at least 50%, 60%, 70% or 80%.
As used herein, xe2x80x9chydrophobicxe2x80x9d amino acid or residue refers to the natural amino acids: methionine, phenylalanine, leucine, isoleucine, or valine.
The amino acid sequence positions described herein, unless otherwise indicated, use as a frame of reference the rOnc sequence of SEQ ID NO:1. Residues labeled with a negative ordinal number indicate the distance from the amino terminus of SEQ ID NO:1 in the direction increasingly distant from the carboxy terminus. It should be understood that position designations do not indicate the number of amino acids in the claimed protein per se, but indicate where in the claimed protein the residue occurs when the claimed protein sequence is aligned with SEQ ID NO:1. The amino acid sequence for SEQ ID NO:1 and for SEQ ID NO:2 are set forth below.
The present invention includes rOnc proteins comprising a polypeptide of SEQ ID NO:1 or conservative variants thereof. The polypeptides of the present invention (SEQ ID NO:1 and conservative variants thereof) demonstrate cytotoxic activity, as defined herein. The rOnc proteins of the present invention may be limited to the polypeptide of SEQ ID NO:1 or conservative variants thereof, or may be inclusive of additional amino acid residues linked via peptide bond to the carboxy and/or amino terminus of the polypeptide. Preferably, the conservative variants of SEQ ID NO:1 comprise an amino terminal glutamine residue capable of spontaneous cyclization to a pyroglutamyl residue.
The polypeptide of SEQ ID NO:1 or conservatively modified variants thereof may have a leucine or other hydrophobic residue substituting for the methionine at position 23. Those of skill will recognize that a polypeptide lacking a methionine is typically not subject to specific cleavage using cyanogen bromide. The polypeptides of the present invention preferably have a lysine at position 9, a histidine at position 10, a lysine at position 31, a threonine at position 35, a histidine at position 97, or a phenylalanine at position 98, or combinations thereof.
Proteins of the present invention can be produced by recombinant expression of a nucleic acid encoding the polypeptide followed by purification using standard techniques. Typically, the rOnc proteins are encoded and expressed as a contiguous chain from a single nucleic acid. The length of the rOnc proteins of the present invention is generally less than about 600 amino acids in length.
Recombinant Onc proteins can also be synthetically prepared in a wide variety of well-known ways. Polypeptides of relatively short size are typically synthesized in solution or on a solid support in accordance with conventional techniques. See, e.g., Merrifield (1963) J. Am. Chem. Soc. 85:2149-2154. Recombinantly produced or synthetic polypeptides can be condensed to form peptide bonds with other polypeptides or proteins formed synthetically or by recombinant methods. Various automatic synthesizers and sequencers are commercially available and can be used in accordance with known protocols. See, e.g., Stewart and Young (1984) Solid Phase Peptide Synthesis, 2d. ed., Pierce Chemical Co.
The present invention also includes rOnc proteins comprising: 1) a polypeptide of SEQ ID NO:1 or conservatively modified variant thereof, and 2) an amino terminal methionine. Isolated nucleic acids coding for the rOnc proteins of the present invention are also provided. Preferably, the amino terminal residue of the polypeptide is a glutamine. Various embodiments of the polypeptide of SEQ ID NO:1 and conservative variants thereof may be employed in this aspect of the invention.
Those of skill will understand that an amino terminal methionine or formlymethionine (collectively, xe2x80x9cmethioninexe2x80x9d) is typically required for protein synthesis in a bacterial host cell. The amino terminal methionine may be directly linked to the amino acid of position 1 of the polypeptides of the present invention via a peptide bond. Alternatively, the methionine is indirectly linked to the amino acid of position 1 of the polypeptides of the present invention via a plurality of peptide bonds from a contiguous chain of amino acid residues. The residues, extending and inclusive of the amino terminal methionine to the amino acid directly linked via a peptide bond to the amino terminal amino acid residue of the polypeptide, constitute an amino terminal peptide. Thus, the amino terminal peptide consists of all amino acid residues with a negative ordinal numbers linked to position +1 of SEQ ID NO:1 or conservatively modified variants thereof and has at its amino terminus a methionine residue. The amino terminal peptide is at least one amino acid residue in length (i.e., a methionine residue) or may be 5, 10, 20, 50, 100, 200, 300, 400, or more amino acids in length.
The amino terminal peptide may comprise, a signal sequence for transport into various organelles or compartments of the host cell, or for transport into the surrounding media. The amino terminal peptide may also encode sequences which aid in purification such as epitopes which allow purification via immunoaffinity chromatography, or sequences recognized by endoproteases such as Factor Xa.
The present invention is also directed to methods of making the rOnc polypeptides of SEQ ID NO:1 or conservative variants thereof. The polypeptides of the SEQ ID NO:1 or conservative variants thereof may conveniently be assayed for cytotoxicity or anti-viral (e.g., HIV-1) inhibition by methods disclosed herein.
A. Expression
The method comprises expressing in a host cell a nucleic acid encoding a polypeptide of SEQ ID NO:1 or conservative variant thereof, where the nucleic acid encodes an amino terminal methionine. Various embodiments of the polypeptides of the present invention previously described may be utilized in this aspect of the invention. By xe2x80x9chost cellxe2x80x9d is meant a cellular recipient, or extract thereof, of an isolated nucleic acid which allows for translation of the nucleic acid and requires an amino terminal methionine for translation of the nucleic acid into its encoded polypeptide. Eukaryotic and prokaryotic host cells may be used such as animal cells, bacteria, fungi, and yeasts. Methods for the use of host cells in expressing isolated nucleic acids are well known to those of skill and may be found, for example, in Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology volume 152 Academic Press, Inc., San Diego, Calif. (Berger); Sambrook et al. (1989) Molecular Cloningxe2x80x94A Laboratory Manual (2nd ed.) Vol. 1-3; and Current Protocols in Molecular Biology, F. M. Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley and Sons, Inc., (1994 Supplement) (Ausubel). A variety of host cells and expression vectors are available from commercial vendors, or the American Type Culture Collection (Rockville, Md.). Accordingly, this invention also provides for host cells and expression vectors comprising the nucleic acid sequences described herein.
Nucleic acids encoding rOnc proteins can be made using standard recombinant or synthetic techniques. Nucleic acids may be RNA, DNA, or hybrids thereof. Given the polypeptides of the present invention, one of skill can construct a variety of clones containing functionally equivalent nucleic acids, such as nucleic acids which encode the same polypeptide. Cloning methodologies to accomplish these ends, and sequencing methods to verify the sequence of nucleic acids are well known in the art. Examples of appropriate cloning and sequencing techniques, and instructions sufficient to direct persons of skill through many cloning exercises are found in Berger and Kimmel; Sambrook et al.; and F. M. Ausubel et al. (all supra). Product information from manufacturers of biological reagents and experimental equipment also provide information useful in known biological methods. Such manufacturers include the SIGMA chemical company (Saint Louis, Mo.), RandD systems (Minneapolis, Minn.), Pharmacia LKB Biotechnology (Piscataway, N.J.), CLONTECH Laboratories, Inc. (Palo Alto, Calif.), Chem Genes Corp., Aldrich Chemical Company (Milwaukee, Wis.), Glen Research, Inc., GIBCO BRL Life Technologies, Inc. (Gaithersberg, Md.), Fluka Chemica-Biochemika Analytika (Fluka Chemie A G, Buchs, Switzerland), Invitrogen, San Diego, Calif., and Applied Biosystems (Foster City, Calif.), as well as many other commercial sources known to one of skill.
The nucleic acid compositions of this invention, whether RNA, cDNA, genomic DNA, or a hybrid of the various combinations, are isolated from biological sources or synthesized in vitro. Deoxynucleotides may be synthesized chemically according to the solid phase phosphoramidite triester method described by Beaucage and Caruthers (1981), Tetrahedron Letts., 22(20):1859-1862, e.g., using an automated synthesizer, e.g., as described in Needham-VanDevanter et al. (1984) Nucleic Acids Res., 12:6159-6168.
One of skill will recognize many ways of generating alterations or variants of a given nucleic acid sequence. Such well-known methods include site-directed mutagenesis, PCR amplification using degenerate oligonucleotides, exposure of cells containing the nucleic acid to mutagenic agents or radiation, chemical synthesis of a desired oligonucleotide (e.g., in conjunction with ligation and/or cloning to generate large nucleic acids) and other well-known techniques. See, Giliman and Smith (1979) Gene 8:81-97, Roberts et al. (1987) Nature 328:731-734 and Sambrook, Innis, Ausubel, and Berger (all supra).
The nucleic acid sequences encoding the rOnc molecules or the fusion proteins may be expressed in a variety of host cells, including E. coli, other bacterial hosts, yeast, and various higher eukaryotic cells such as the COS, CHO and HeLa cells lines and myeloma cell lines. The recombinant nucleic acid will be operably linked to appropriate expression control sequences for each host. For E. coli this includes a promoter such as the T7, trp, or lambda promoters, a ribosome binding site and preferably a transcription termination signal. For eukaryotic cells, the control sequences will include a promoter and preferably an enhancer derived from immunoglobulin genes, SV40, cytomegalovirus, etc., and a polyadenylation sequence, and may include splice donor and acceptor sequences.
The expression vectors or plasmids of the invention can be transferred into the chosen host cell by well-known methods such as calcium chloride transformation for E. coli and calcium phosphate treatment, liposomal fusion or electroporation for mammalian cells. Cells transformed by the plasmids can be selected by resistance to antibiotics conferred by genes contained on the plasmids, such as the amp, gpt, neo and hyg genes.
Once expressed, the rOnc protein can be purified according to standard procedures of the art, including ammonium sulfate precipitation, column chromatography (including affinity chromatography), gel electrophoresis and the like (see, generally, R. Scopes, Protein Purification, Springer-Verlag, N.Y. (1982), Deutscher, Methods in Enzymology Vol. 182: Guide to Protein Purification., Academic Press, Inc. N.Y. (1990)).
B. Cleaving
After expression in the host cell, the resultant rOnc protein comprising an amino terminal methionine is treated with a cleaving agent or combination of cleaving agents. By xe2x80x9ccleaving the amino terminal methioninexe2x80x9d is meant cleaving the amino terminal methionine or amino terminal peptide from the polypeptides of SEQ ID NO:1 or conservative variants thereof. Thus, by xe2x80x9ccleaving the amino terminal methioninexe2x80x9d, a polypeptide of SEQ ID NO:1 or conservative variant thereof is generated, optionally linked via peptide bonds to additional residues at the carboxy terminus.
The cleaving agent may be a proteolytic enzyme such as an exopeptidase or endopeptidase (collectively, xe2x80x9cpeptidasexe2x80x9d) or a chemical cleaving agent. Exopeptidases include aminopeptidase M (Pierce, Rockford, Ill.) which sequentially remove amino acids from the amino-terminus. Cleavage of the amino terminal methionine by exopeptidases may be controlled by modulating the enzyme concentration, temperature, or time under which the cleavage takes place. The resulting mixture may be purified for the desired protein by means well known to those of skill, for example, on the basis of length by electrophoresis. Endopeptidases useful for removing the amino terminal methionine and other residues of the amino terminal peptide include Factor Xa (Pierce) which cleaves at the carboxy side of Ile-Glu-Gly-Arg (SEQ ID NO:3) sequence. The chemical cleaving agent, cyanogen bromide, is conveniently employed to selectively cleave methionine residues.
The cleaving agent employed to cleave the amino terminal methionine will typically be chosen so as not to break a peptide bond within the polypeptide of SEQ ID NO:1 or conservative variants thereof. Alternatively, use of a particular cleaving agent may guide the choice of conservative substitutions of the conservative variants of the polypeptides of the present invention.
C. Cyclization
Upon cleavage of the amino terminal methionine and other residues of the amino terminal peptide, a protein comprising the polypeptide of SEQ ID NO:1 or a conservatively modified variant thereof is generated. The glutamine residue of SEQ ID NO:1 is caused to cyclize by any number of means, including spontaneously or by catalysis, to a pyroglutamyl residue. Spontaneous hydrolysis of amino terminal glutamine residues to their pyroglutamyl form is well known to the skilled artisan and its rate may be hastened by, for example, increasing the temperature. See, e.g., Robinson et al., J. Am. Chem. Soc., 95:8156-8159 (1973). Cytotoxicity or anti-viral activity of the resultant rOnc protein may be assessed by means herein disclosed and well known to the skilled artisan.
The polypeptides and proteins of the present invention may also be joined via covalent or non-covalent bond to a ligand binding moiety The rOnc molecule may be joined at the carboxy terminus to the ligand or may also be joined at an internal region as long as the attachment does not interfere with the respective activities of the molecules. Immunoglobulins or binding fragments-thereof (e.g., single-chain Fv fragments) may conveniently be joined to the polypeptides of the present invention. Vaughan et al., Nature Biotechnology, 14:309-314 (1996).
The molecules may be attached by any of a number of means well-known to those of skill in the art. Typically the rOnc protein will be conjugated, either directly or through a linker (spacer), to the ligand. However, where both the rOnc and the ligand or other therapeutic are polypeptides it is preferable to recombinantly express the chimeric molecule as a single-chain fusion protein.
The procedure for attaching an agent to an antibody or other polypeptide targeting molecule will vary according to the chemical structure of the agent. Polypeptides typically contain a variety of functional groups; e.g., carboxylic acid (COOH) or free amine (xe2x80x94NH2) groups, which are available for reaction with a suitable functional group on an rOnc molecule to bind the other molecule thereto.
Alternatively, the ligand and/or rOnc molecule may be derivatized to expose or attach additional reactive functional groups. The derivatization may involve attachment of any of a number of linker molecules such as those available from Pierce Chemical Company, Rockford Ill.
A xe2x80x9clinkerxe2x80x9d, as used herein, is a molecule that is used to join two molecules. The linker is capable of forming covalent bonds to both molecules. Suitable linkers are well known to those of skill in the art and include, but are not limited to, straighter branched-chain carbon linkers, heterocyclic carbon linkers, or peptide linkers. Where both molecules are polypeptides, the linkers may be joined to the constituent amino acids through their side groups (e.g., through a disulfide linkage to cysteine).
A bifunctional linker having one functional group reactive with a group on a particular agent, and another group reactive with an antibody, may be used to form a desired immunoconjugate. Alternatively, derivatization may involve chemical treatment of the ligand, e.g., glycol cleavage of the sugar moiety of a glycoprotein antibody with periodate to generate free aldehyde groups. The free aldehyde groups on the antibody may be reacted with free amine or hydrazine groups on an agent to bind the agent thereto. (See U.S. Pat. No. 4,671,958). Procedures for generation of free sulfhydryl groups on polypeptides, such as antibodies or antibody fragments, are also known (See U.S. Pat. No. 4,659,839).
Many procedures and linker molecules for attachment of various compounds including radionuclide metal chelates, toxins and drugs to proteins such as antibodies are known. See, for example, European Patent Application No. 188,256; U.S. Pat. Nos. 4,671,958, 4,659,839, 4,414,148, 4,699,784; 4,680,338; 4,569,789; 4,589,071; and Borlinghaus et al. Cancer Res. 47: 4071-4075(1987), which are incorporated herein by reference. In particular, production of various immunotoxins is well-known within the art and can be found, for example in xe2x80x9cMonoclonal Antibody-Toxin Conjugates: Aiming the Magic Bullet,xe2x80x9d Thorpe et al., Monoclonal Antibodies in Clinical Medicine, Academic Press, pp. 168-190 (1982), Waldmann, Science, 252: 1657 (1991), U.S. Pat. Nos. 4,545,985 and 4,894,443 which are incorporated herein by reference.
In some circumstances, it is desirable to free the rOnc from the ligand when the chimeric molecule has reached its target site. Therefore, chimeric conjugates comprising linkages which are cleavable in the vicinity of the target site may be used when the effector is to be released at the target site. Cleaving of the linkage to release the agent from the ligand may be prompted by enzymatic activity or conditions to which the immunoconjugate is subjected either inside the target cell or in the vicinity of the target site. When the target site is a tumor, a linker which is cleavable under conditions present at the tumor site (e.g. when exposed to tumor-associated enzymes or acidic pH) may be used.
A number of different cleavable linkers are known to those of skill in the art. See U.S. Pat. Nos. 4,618,492; 4,542,225, and 4,625,014. The mechanisms for release of an agent from these linker groups include, for example, irradiation of a photolabile bond and acid-catalyzed hydrolysis. U.S. Pat. No. 4,671,958, for example, includes a description of immunoconjugates comprising linkers which are cleaved at the target site in vivo by the proteolytic enzymes of the patient""s complement system. In view of the large number of methods that have been reported for attaching a variety of radiodiagnostic compounds, radiotherapeutic compounds, drugs, toxins, and other agents to antibodies one skilled in the art will be able to determine a suitable method for attaching a given agent to an antibody or other polypeptide.